Synthetic gene clusters

Patent metadata

Field	Value
Publication number	US-9512431-B2
Application number	US-201214126307-A
Country	US
Kind code	B2
Filing date	Jun 14, 2012
Priority date	Jun 16, 2011
Publication date	Dec 6, 2016
Grant date	Dec 6, 2016

Abstract

Methods for making synthetic gene clusters are described.

First claim

What is claimed is:

1. A method for expressing one or more synthetic operons collectively associated with a function in a cell by replacing native regulation of a set of genes with synthetic regulation, the method comprising:
providing coding sequences for a set of polypeptides encoded by genes collectively associated with a function;
changing codon identity within at least one coding sequence, thereby removing at least one regulatory sequence within the coding sequence, wherein removing the at least one regulatory sequence comprises replacement of native codons in the coding sequence with non-native synonymous codons and comprises selecting non-native codons having maximal distance from the native codons of the coding sequence;
organizing the coding sequences into one or more synthetic operon(s);
operably linking one or more heterologous transcriptional regulatory sequence to the operon(s), thereby controlling magnitude of gene expression from the operon(s); and
expressing the one or more synthetic operon(s) in a cell under the control of a polypeptide that binds directly or indirectly to the heterologous transcriptional regulatory sequence,
wherein the polypeptide that binds directly or indirectly to the heterologous transcriptional regulatory sequence is expressed from a control expression cassette, the expression cassette comprising a control promoter operably linked to a polynucleotide sequence encoding the polypeptide.

2. The method of claim 1 , wherein the polypeptide that binds directly or indirectly to the heterologous transcriptional regulatory sequence is heterologous to the cell.

3. The method of claim 1 , wherein the providing comprises obtaining the gene nucleotide sequences and eliminating non-coding sequences.

4. The method of claim 1 , wherein the genes are from a gene cluster.

5. The method of claim 1 , wherein the genes are from a prokaryote.

6. The method of claim 5 , wherein the genes are from a native operon.

7. The method of claim 1 , wherein the at least one regulatory sequence is identified using computation.

8. The method of claim 7 , wherein the computation comprises searches of coding sequences for ribosome binding sites, terminators, and/or promoters.

9. The method of claim 1 , further comprising identifying and removing one or more of transposon insertion sites, sites that promote recombination, sites for cleavage by restriction endonucleases, and sites that are methylated.

10. The method of claim 1 , wherein the organizing comprises grouping coding sequences into operons based on substantially similar native expression level.

11. The method of claim 1 , wherein the organizing comprises ordering coding sequences within operons such that the highest expressing gene (based on native expression) occurs first and the lowest expressing gene (based on native expression) occurs last.

12. The method of claim 1 , wherein the heterologous transcriptional regulatory sequence(s) comprise a T7 RNAP promoter(s).

13. The method of claim 1 , wherein heterologous transcriptional regulatory sequence(s) comprise an inducible promoter.

14. The method of claim 1 , further comprising operably linking a heterologous transcriptional terminator sequence to one or more coding sequence in the synthetic operon.

15. The method of claim 14 , wherein the heterologous transcriptional terminator sequence is a T7 RNAP terminator.

16. The method of claim 14 , wherein heterologous transcriptional terminator sequences for different operons are different.

17. The method of claim 1 , wherein the operon(s) are expressed from a plasmid.

18. The method of claim 17 , wherein the plasmid has a low copy origin of replication.

19. The method of claim 1 , wherein the expression cassette is contained in a control plasmid separate from a plasmid containing the operon(s).

20. The method of claim 1 , wherein the control promoter is an inducible promoter.

21. The method of claim 1 , wherein the heterologous polypeptide comprises an RNA polymerase (RNAP).

22. The method of claim 21 , wherein the RNAP is T7 RNAP.

23. The method of claim 1 , wherein the expression cassette is an environmental sensor.

24. The method of claim 1 ,
wherein relative magnitude of expression of coding sequences in the synthetic operon(s) substantially corresponds to relative protein levels in the native system.

25. A method for expressing one or more synthetic operons collectively associated with a function in a cell by replacing native regulation of a set of genes with synthetic regulation, the method comprising:
providing coding sequences for a set of polypeptides encoded by genes collectively associated with a function;
changing codon identity within at least one coding sequence by removing at least one regulatory sequence within the coding sequence, wherein removing the at least one regulatory sequence comprises replacement of native codons in the coding sequence with non-native synonymous codons and comprises selecting non-native codons having maximal distance from the native codons of the coding sequence;
organizing the coding sequences into one or more synthetic operon(s);
operably linking one or more heterologous transcriptional regulatory sequence to the operon(s), thereby controlling magnitude of gene expression from the operon(s); and
expressing the one or more synthetic operon(s) in a cell under the control of a polypeptide that binds directly or indirectly to the heterologous transcriptional regulatory sequence; and
detecting the magnitude of gene expression by computation, wherein the computation comprises a numerical optimization algorithm, and wherein the numerical optimization algorithm comprises the Nelder-Mead algorithm, the Newton's method, the quasi-Newton method, a conjugate gradient method, an interior point method, a gradient descent, a subgradient method, a ellipsoid method, the Frank-Wolfe method, an interpolation method and pattern search methods, or an ant colony model.

26. A method for expressing one or more synthetic operons collectively associated with a function in a cell by replacing native regulation of a set of genes with synthetic regulation, the method comprising:
providing coding sequences for a set of polypeptides encoded by genes collectively associated with a function;
changing codon identity within at least one coding sequence by removing at least one regulatory sequence within the coding sequence, wherein removing the at least one regulatory sequence comprises replacement of native codons in the coding sequence with non-native synonymous codons and comprises selecting non-native codons having maximal distance from the native codons of the coding sequence;
organizing the coding sequences into one or more synthetic operon(s);
operably linking one or more heterologous transcriptional regulatory sequence to the operon(s), thereby controlling magnitude of gene expression from the operon(s);
operably linking a heterologous ribosomal binding site (RBS) to one or more coding sequence in the synthetic operon, wherein different RBSs are operably linked to different coding sequences, and wherein the RBSs regulate translation of the coding sequences in a ratio that is substantially similar to a ratio of translation from a native operon, and
expressing the one or more synthetic operon(s) in a cell under the control of a polypeptide that binds directly or indirectly to the heterologous transcriptional

Assignees

• Mirsky Ethan
• Temme Karsten
• Voigt Christopher A
• Zhao Dehua
• Univ California

Inventors

• Mirsky Ethan
• Temme Karsten
• Voigt Christopher A
• Zhao Dehua

Classifications

• C12N15/74

  Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora · CPC title

• C12N15/63 Primary

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C12N9/0095

  acting on iron-sulfur proteins as donor (1.18) · CPC title

• G16B30/00

  ICT specially adapted for sequence analysis involving nucleotides or amino acids · CPC title

• C12N15/67

  General methods for enhancing the expression · CPC title